This invention relates in general to novel polynucleotides isolated from cDNA libraries of human fetal liver-spleen and macrophages and to polypeptides encoded by these polynucleotides. In particular, the invention relates to a human CD39-like protein with homologies to ATP diphosphohydrolases and variants thereof.
CD39 (cluster of differentiation 39) is a cell-surface molecule recognized by a xe2x80x9cclusterxe2x80x9d of monoclonal antibodies that can be used to identify the lineage or stage of differentiation of lymphocytes and thus to distinguish one class of lymphocytes from another. This CD39 molecule was originally defined as a B lymphocyte marker (Rowe, M., et al. Int. J. Cancer 29:373 (1982)). Subsequent studies have shown CD39 to be a marker for a distinct subset of activated lymphocytes within the allosensitized CD8-positive cytotoxic cells (Gouttefangeas C., et al., Eur. J.Immunol. 22:2681 (1992)). Outside of lymphoid tissue, CD39 can be found in quiescent vascular endothelial cells (Kansas, G. S., et al., J. Immunol. 146:2235 (1991)) and throughout rat brain in the neurons of the cerebral cortex, hippocampus, and cerebellum, as well as in glial cells (Wang, T-F. and Guidotti, G., Brain Res. 790:318 (1998)).
CD39 is a 510-amino acid protein with a predicted molecular mass of 57 kDa. However, because of heavy glycosylation at asparagine residues (six potential N-glycosylation sites) the molecule displays a mobility closer to 100 kDa (Maliszewski, C. R., et al., J. Immunol. 153:3574 (1994)). CD39 contains two hydrophobic regions, one near the amino terminus and the other near the carboxyl terminus which are believed to be transmembrane regions.
The role of CD39 in platelet aggregation and ATP/ADP hydrolysis is unclear. Although CD39 was originally reported to be an ectoADPase with a preference for ATP over ADP as a substrate, Wang, et al., J. Biol. Chem. 271:9898-9901(1996), Marcus, et al., J. Clin. Invest. 99:1351-1360 (1997) reported that CD39 was unique for its high preference for ADP over ATP as a substrate and in 1998, Gayle, et al., J. Clin Invest. 10:1851-1859 (1998), described CD39 as an ectoADPase with no preference for one substrate over the other.
Reports that several ATP Diphosphohydrolases (ATPDases) share amino acid sequence homology with CD39 have been substantiated by the showing that CD39 is itself an ATPDase (Wang, T-F., et al., J. Biol. Chem. 271:9898 (1996); Kaczmarek, E., et al., J. Biol. Chem. 271:33116 (1996)). Since CD39 is a plasma membrane-bound enzyme, CD39 has been termed an xe2x80x9cecto-ATPase,xe2x80x9d but CD39 is more often referred to as an xe2x80x9cecto-apyrasexe2x80x9d because of the reduced rate of hydrolysis of ADP when compared with ecto-ATPases.
This activity has shown to modulate platelet reactivity and aggregation in response to vascular injury. During vascular injury, activated platelets aggregate forming an occlusive thrombus. Excessive platelet accumulation at sites of vascular injury can contribute to vessel occlusion. Endothelial cells respond to the potentially occlusive effects of platelet aggregation by several mechanisms. One of these mechanisms results ecto-apyrase-mediated removal of ADP, which in turn eliminates platelet reactivity and recruitment. It is now known that the endothelial ecto-apyrase responsible for this ADP removal is CD39 (Marcus, A. J., et al., J. Clin. Invest. 99:1351 (1997)).
Recently, CD39 was engineered to produce a soluble form of the molecule. This soluble CD39 was shown to display the same nucleotidase activity as the membrane-bound molecule (Gayle, R. B., et al., J. Clin. Invest. 101:1851 (1998)). Intravenously administered soluble CD39 also remained active in mice for an extensive period of time, indicating that soluble CD39 could be useful as a inhibitor of platelet aggregation in the prophylaxis or treatment of platelet-mediated thrombotic conditions.
Platelet aggregation inhibitors (antithrombotic agents) decrease the formation or the action of chemical signals that promote platelet aggregation. Currently available antithrombotic agents include aspirin, ticlopidine, and dipyridamole. These agents have proven beneficial in the prevention and treatment of occlusive cardiovascular diseases, including myocardial infarction, cerebral ischemia, angina. Antithrombotic therapy has also been used in the maintenance of vascular grafts.
Myocardial infarction is the development of necrosis of the myocardium (the middle muscular layer of the heart wall) due to a critical imbalance between oxygen and myocardial demand. The most common cause of acute myocardium infarction is narrowing of the epicardial blood vessels due to atheromatous plaques. Plaque rupture with subsequent exposure of basement membrane results in platelet aggregation and thrombus formation, which can result in partial or complete occlusion of the vessel and subsequent myocardial ischemia.
In cerebral ischemia, inadequate blood flow results from an occlusion in a blood vessel or hemorrhaging. In the latter case, excessive bleeding in one area of the brain deprives another area of blood. If the damage occurs in a singular small area, xe2x80x9ctransientxe2x80x9d or xe2x80x9cfocusedxe2x80x9d cerebral ischemia results. When a major artery is blocked (carotid artery) global or diffused ischemia results. The primary medical strategy for secondary prevention of stroke is antiplatelet therapy. Aspirin is currently employed for reducing the risk of recurrent transient ischemic attacks or stroke in men who have transient ischemia of the brain due to fibrin emboli.
Each year, thousands of patients suffer a decline in blood flow to one or more limbs. Without sufficient blood flow, and, unless blood flow can be restored in time, the limb must be amputated. In some cases, grafts from the patient""s veins can be used to form new arteries. However, in cases where the quality of the veins is poor, polymeric vascular grafts are typically used. The polymeric grafts are inherently thrombogenic as the blood constituents passing through the grafts become activated and tend to form clots. Efforts to line the grafts with endothelial cells can reduce blood clotting, but better results are obtained when antithrombotic therapy is employed.
Angina pectoris is a characteristic chest pain caused by inadequate blood flow through the blood vessels of the myocardium. The imbalance between oxygen delivery and utilization may result from a spasm of the vascular smooth muscle or from obstruction of blood vessels caused by atherosclerotic lesions. Three classes of drugs have been shown to be effective in treating angina: nitrates, beta-blockers and calcium channel blockers. Currently, the antithrombotics dipyridamole and aspirin are employed to prophylactically treat angina pectoris.
Ecto-apyrases, such as CD39, offer a number of advantages over several of the standard antithrombotics. For example, aspirin treatment controls the prothrombotic action of thromboxane; however, aspirin also prevents formation of antithrombotic prostacyclin, which limits aspirin""s efficacy. Another antithrombotic, endothelium-derived relaxing factor (nitric oxide; xe2x80x9cEDRF/NOxe2x80x9d), is inhibited in vitro and in vivo by hemoglobin after its rapid diffusion into erythrocytes. In contrast, CD39 is aspirin-insensitive and completely inhibits platelet reactivity even when eicosanoid and EDRF/NO production are blocked.
CD39""s ATPDase activity also implicates CD39 in the modulation of neurotransmission. ATP is a major purinergic neurotransmitter that is often co-released into the synaptic cleft with several neurotransmitters. Responses to ATP are mediated by specific plasma membrane receptors, called P2 purinergic receptors (Dubyak, G. R. and El-Motassim,C. Am J. Physiol. 34:C577-C606 (1993)). The distribution of CD39 in the rat brain indicates that CD39 plays a role in terminating P2 purinergic neurotransmission (Wang, T. F. and Guidotti, G., Brain Res. 790:318 (1998)). Furthermore, a decrease in ecto-apyrase activity is believed to lead to an accumulation of the excitatory neurotransmitter, extracellular ATP, as well as a deficiency of the endogenous anticonvulsant extracellular adenosine.
The chomosomal localization of CD39 provides additional support for a role in modulation of neurotransmission. More specifically, CD39 has been mapped to chromosome 10q 23.1-24.1 (Maliszewski, C. R., et al., J. Immunol. 153:3574 (1994)), and this site overlaps with the susceptibility locus for human partial epilepsy with audiogenic symptoms (Ottman, R. et al., Nature Genet. 10:56 (1995)). This co-localization of the CD39 gene and the susceptibility locus has led to the hypothesis that decrease in ecto-apyrase activity in the brain is the primary cause of partial epilepsy (Wang T-F., et al., Mol. Brain Res. 47:295 (1997)).
A screen for human cDNAs that hybridize to cosmids from the human chromosome 9q34 region lead to the identification of a transcript with high homology to a chicken muscle ecto-ATPase (60% identity) and the ecto-apyrase CD39 (41% amino acid identity) (Chadwick, B. P., Mamm. Genome 8:668 (1997)). This gene, designated xe2x80x9cCD39-like-1 genexe2x80x9d (CD39L1), has a higher degree of homology to CD39 than does chicken muscle ecto-ATPase. The biological activity of this protein has not been tested but on the basis of the high amino acid homology, CD39L1 is believed to be a new member of the ecto-ATPase family. Recently, a mouse gene with homology to NTPases was cloned and sequenced (Acc. No. AF006482) by Chadwick et al. (Mamm. Gen. 9:162-164 (1998).)
The invention is based on polynucleotides isolated from cDNA libraries prepared from human fetal liver-spleen and macrophages. The compositions of the present invention include novel isolated polypeptides with apyrase and/or NDPase activity, in particular, novel human CD39-like polypeptides, and active variants thereof, isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.
The compositions of the invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.
The isolated polynucleotides of the invention include naturally occurring or wholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA. One polynucleotide according to the invention encodes a novel CD39-like protein having the amino acid sequence shown in FIG. 2 (SEQ ID NO: 3), which has been designated CD39-L4. Another polynucleotide according to the invention encodes a novel CD39-like protein having the amino acid sequence shown in SEQ ID NO: 27, which has been designated CD39-L2. In another embodiment, a polynucleotide according to the invention encodes a novel CD39-like protein having the full length or mature amino acid sequence set forth in SEQ ID NO: 25, which has been designated CD39-L66, and is an isoform of CD39-L4. The isolated polynucleotides of the invention include a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2, 24 or 26. The polynucleotides of the invention also include polynucleotides that encode polypeptides with a biological activity of the polypeptide of SEQ ID NO: 3 or 27 (including apyrase or NDPase activity) such as (a) the nucleotide sequence of SEQ ID NO: 2, 24, 26 or (b) a nucleotide sequence encoding the full length or mature amino acid sequence of SEQ ID NO: 3, 25, or 27; (c) a polynucleotide which is an allelic variant of any polynucleotide recited above; (d) a polynucleotide that hybridizes under stringent conditions to (a) or (b); (e) or a polynucleotide that encodes a polypeptide comprising at least one CD39-like domain, e.g. catalytic domain.
The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above.
A collection as used in this application can be a collection of only one polynucleotide. The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array. In one embodiment, segments of sequence information are provided on a nucleic acid array to detect the polynucleotide that contains the segment. The array can be designed to detect nucleic acids that are perfectly complementary (full-match) or mismatched to the polynucleotide that contains the segment. The collection can also be provided in a computer-readable format.
The invention also provides a polynucleotide including a nucleotide sequence that is substantially equivalent to these polynucleotides. Polynucleotides according to the invention can have at least about 80%, more typically at least about 90%, and even more typically at least about 95%, sequence identity to a polynucleotide of SEQ ID NO: 2, 24 or 26 and specifically include a human polynucleotide which has at least 80% sequence identity to a polynucleotide of SEQ ID NO: 2, 24 or 26 ; or a polynucleotide which has at least 90% sequence identity to a polynucleotide of SEQ ID NO: 2, 24 or 26. Similarly, polypeptides of the invention include polypeptides having apyrase or NDPase activity and at least about 80%, 90% or 95% sequence identity to SEQ ID NO: 3, 25 or 27. Polypeptides of the invention further include multimeric, especially dimeric, polypeptides having apyrase or NDPase activity and at least about 80%, 90% or 95% sequence identity to SEQ ID NO: 3, 25 or 27.
A further aspect of the invention is the development of novel CD39-L4 polynucleotide or polypeptide variants which preferably exhibit increased recombinant expression levels or improved ADPase or NDPase activity compared to wild type CD39-L4 (SEQ ID NO: 5). This aspect of the invention includes polypeptides comprising at least one amino acid substitution selected from the group consisting of: D168xe2x86x92T, S170xe2x86x92Q and L175xe2x86x92F, wherein said substitution(s) result in increased ADPase activity of the polypeptide. One preferred embodiment is the polypeptide having the amino acid sequence set forth in SEQ ID NO: 7 (encoded by the nucleotide sequence of SEQ ID NO: 6), which is a variant CD39-L4 containing all three substitutions that has been designated ACRIII. A plasmid containing this DNA was deposited with the American Type Culture Collection (ATCC), 10801 University Avenue, Manassas, Va., on Jul. 13, 1999 under the terms of the Budapest Treaty (ATCC accession number PTA-346). Alternatively, instead of making the specific D168xe2x86x92T, S170xe2x86x92Q and/or L175xe2x86x92F substitution(s), substitution of amino acids with similar properties is contemplated. Additional conservative substitutions at amino acid positions other than D168, S170 and/or L175 are further contemplated. For example, all of the corresponding amino acids from CD39 could be substituted for amino acids 167-181 of CD39-L66 or CD39-L4.
This aspect of the invention also specifically contemplates that, in view of the fact that variant polynucleotides containing changes in the codons for amino acid 168, 170 and 175 are more highly expressed, such polynucleotides maybe more highly expressed if the codons at these same positions are modified without changing the wild type amino acid sequence (e.g., polynucleotides having codon substitutions at or around nucleotide positions 747-749, 753-755, and/or 768-770 of SEQ ID NO: 2, or positions 502-504, 508-510 and/or 523-525 of SEQ ID NO: 4, or corresponding nucleotide positions in SEQ ID NO: 24 are contemplated).
In addition, development of novel CD39-L2 polynucleotide or polypeptide variants which preferably exhibit increased recombinant expression levels or improved ADPase or NDPase activity compared to wild type CD39-L2 (SEQ ID NO: 27) is also contemplated. This aspect of the invention includes polypeptides comprising at least one amino acid substitution wherein said substitution(s) result in increased recombinant expression levels or increased ADPase activity of the polypeptide, as well as polynucleotides encoding the wild type CD39-L2 sequence that have silent codon substitutions at nucleotide positions 806-808, 812-814 and/or 827-829 corresponding to those identified above for CD39-L4.
Also provided by the invention are CD39L4 polypeptides with apyrase and/or NDPase activity, comprising the mature protein amino acid sequence of SEQ ID NO: 3, wherein the cysteine at position 39 (Cys39) of SEQ ID NO: 3 is deleted or replaced with a different amino acid, preferably a neutral amino acid. CD39L4 variants containing additional mutations (e.g. conservative substitutions) and wherein a cysteine corresponding to Cys39 of SEQ ID NO: 3 is deleted or replaced are also contemplated. Recombinantly-expressed preparations of these cysteine-mutants exhibit increased activity due to reduced formation of inactive dimers.
Polynucleotides encoding these polypeptides, vectors and host cells comprising such polynucleotides, methods of using such host cells to produce polypeptides, and other therapeutic products comprising the polypeptides (including fusion proteins in which the CD39-like polypeptide is fused to a heterologous peptide or polypeptide, such as an immunoglobulin constant region, or derivatives in which the CD39-like polypeptide is modified by water soluble polymers to increase its half-life) are also comprehended by the invention, as are methods of treating a subject suffering from a disorder relating to thrombosis, coagulation or platelet aggregation by administering such therapeutic products.
The invention further comprises methods of inhibiting platelet aggregation in a mammalian subject by reducing the ratio of ADP:ATP in a mammalian subject to a less than normal ratio by administering the polypeptides of the invention or by administering polypeptides with ADPase activty and at least about 90% sequence identity to SEQ ID NO: 3, 25 or 27. Preferably the ratio of ADP:ATP is reduced without significantly affecting ATP levels. In one embodiment, the ADP:ATP ratio is reduced systemically in circulation. In another embodiment, the ADP:ATP ratio is reduced locally, for example, in heart, brain, kidney, lungs, limbs or other organs.
Methods of identifying compounds capable of reducing the ratio of ADP:ATP to a less than normal ratio are also contemplated. For example, compounds may be identified by steps including: determining apyrase activity of said compound on ATP; determining apyrase activity of said compounds on ADP; and selecting a compound that has greater activity with respect to ADP compared to ATP. Exemplary compounds to be screened include, but are not limited to, CD39-L4 and CD39-L2 variants.
Gene therapy techniques are also provided to modulate disease states associated with CD39-L4 or CD39-L2 expression and/or biological activity. Delivery of a functional CD39-L4 or CD39-L2 gene to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments).
The invention also relates to methods for producing polypeptides of the invention comprising growing a culture of cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the protein from the cells or the culture medium. Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.
Protein compositions of the present invention, including therapeutic compositions, comprise polypeptides of the invention and optionally an acceptable carrier, such as a hydrophilic (e.g., pharmaceutically acceptable) carrier.
Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use as oligomers for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like. For example, because the expression of CD39-L4 and CD39-L2 mRNA is largely restricted to specific tissues (CD39-L4 in macrophages and CD39-L2 in adult heart and fetal brain), polynucleotides of the invention can be used as hybridization probes to detect the presence of specific mRNA in a sample using, e.g., in situ hybridization.
In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath, et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.
A polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook, J., et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Useful nucleotide sequences for joining to polypeptides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art. Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide. In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell. Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.
The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins. For example, a polypeptide of the invention can be used to generate an antibody which specifically binds the polypeptide. The polypeptides of the invention having ATPDase activity are also useful for inhibiting platelet aggregation and can therefore be employed in the prophylaxis or treatment of pathological conditions caused by the inflammatory response. The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.
Another aspect of the invention is an antibody that specifically binds the polypeptide of the invention. Such antibodies can be either monoclonal or polyclonal antibodies, as well fragments thereof and humanized forms or fully human forms, such as those produced in transgenic animals. The invention further provides a hybridoma that produces an antibody according to the invention and anti-idiotype antibodies.
Antibodies of the invention are useful for detection and/or purification of the polypeptides of the invention.
Methods are also provided for preventing, treating or ameliorating a medical condition, including thrombotic diseases, which comprises administering to a mammalian subject, including but not limited to humans, a therapeutically effective amount of a composition comprising a polypeptide of the invention or a therapeutically effective amount of a composition comprising a binding partner of (e.g., antibody specifically reactive for) CD39-like polypeptides of the invention. The mechanics of the particular condition or pathology will dictate whether the polypeptides of the invention or binding partners (or inhibitors) of these would be beneficial to the individual in need of treatment.
The invention also provides a method of inhibiting platelet function comprising administering a CD39-L4 or CD39-L2 polypeptide of the invention to a medium comprising platelets. According to this method, polypeptides of the invention can be administered to produce an in vitro or in vivo inhibition of platelet function. A polypeptide of the invention can be administered in vivo as antithrombotic agent alone or as an adjunct to other therapies.
Also provided are methods of hydrolyzing nucleotide diphosphates comprising administering CD39-L4 or CD39-L2 polypeptides of the invention to a medium comprising nucleotidediphosphates. According to this method, polypeptides of the invention can be administered to produce an in vitro or in vivo hydrolysis of nucleotidediphosphates. A polypeptide of the invention can be administered in vivo alone or as an adjunct to other therapies. For example, CD39-L4 or CD39-L2 polypeptides of the invention may be administered to prevent or treat cancer conditions involving elevated levels of one or more nucleotide diphosphates.
The invention further provides methods for manufacturing medicaments useful in the above described methods relating to platelet aggregation and thrombosis.
The invention also provides methods for detecting or quantitating the presence of the polynucleotides or polypeptides of the invention in a tissue or fluid sample, and corresponding kits that comprise suitable polynucleotide probes or antibodies, together with an optional quantitative standard. Such methods and kits can be utilized as part of prognostic and diagnostic evaluation of patients and for the identification of subjects exhibiting a predisposition to platelet mediated conditions.
The invention also provides methods for the identification of compounds that modulate (i.e. increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention. Such methods can be utilized, for example, for the identification of compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention, and assays for identifying compounds and other substances that enhance or inhibit the activity of the polypeptides of the invention, such assays comprising the step of measuring activity of such polypeptides in the presence and absence of the test compound.